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Objective
- To run electrophoresis(SDS-PAGE) and UV vis on the pepsin and pepstatin prepared on 09/24/13.
Protocol
General Protocol:
- Prepare the gel and assemble the electrophoresis cell
- Refer here for exact details on how to assemble the unit.
- Remove comb and tape from the gel
- Rinse the walls with running buffer (30.0 Tris, 44.1g glycine, 10g SDS, H2O to 1L)
- Assemble cell
- Fill the inner and outer buffer chambers with running buffer
- Load samples prepared yesterday
- Note: Also loaded two hemeglobin samples for comparison and pepsin and pepstatin reactions left overnight(these were prepared in the same fashion as 2013/09/24. In total ran 12 samples.
- Note: Loaded entire 20μL samples
- Run electrophoresis for 30 minutes at 200V
- Develop stain of the gel
- Place gel in fixant solution (40% methanol, 10% acetic acid, 50% water) for 20 minutes to stop the proteins from migrating further
- Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
- Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
- Repeat last part of step 4 twice.
Sample Set-up of the electrophoresis (SDS-PAGE)
| Well Numbers
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Sample
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| 1 |
Hemoglobin
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| 2 |
Pepsin
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| 3 |
Pepstatin
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| 4 |
0.5hrs Pepsin
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| 5 |
0.5 hrs Pepstatin
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| 6 |
1 hr Pepsin
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| 7 |
1 hr Pepstatin
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| 8 |
1.5hrs Pepsin
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| 9 |
1.5 hrs Pepstatin
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| 10 |
2hrs Pepsin
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| 11 |
2hrs Pepstatin
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| 12 |
Hemoglobin
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UV vis
Corrected Absorbance Spectra of Pepsin with Hemoglobin
NOTES: For the 2 hour sample some pieces of protein may have been present when running the sample.
Corrected Absorbance Spectra of 20μM Pepstatin with Hemoglobin
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